Grazia Chiellini

The Thyroid Hormone Receptor-β-Selective Agonist GC-1 Differentially Affects Plasma Lipids and Cardiac Activity1

Thyroid hormones influence the function of many organs and mediate their diverse actions through two types of thyroid hormone receptors, TRα and TRβ. Little is known about effects of ligands that preferentially interact with the two different TR subtypes. In the current study the comparison of the effects of the novel synthetic TRβ-selective compound GC-1 with T3 at equimolar doses in hypothyroid mice revealed that GC-1 had better triglyceride-lowering and similar cholesterol-lowering effects than T3. T3, but not GC-1, increased heart rate and elevated messenger RNA levels coding for the If channel (HCN2), a cardiac pacemaker that was decreased in hypothyroid mice. T3 had a larger positive inotropic effect than GC-1. T3, but not GC-1, normalized heart and body weights and messenger RNAs of myosin heavy chain α and β and the sarcoplasmic reticulum adenosine triphosphatase (Serca2). Additional dose-response studies in hypercholesteremic rats confirmed the preferential effect of GC-1 on TRβ-mediated paramete...

Ligand selectivity by seeking hydrophobicity in thyroid hormone receptor

Selective therapeutics for nuclear receptors would revolutionize treatment for endocrine disease. Specific control of nuclear receptor activity is challenging because the internal cavities that bind hormones can be virtually identical. Only one highly selective hormone analog is known for the thyroid receptor, GC-24, an agonist for human thyroid hormone receptor β. The compound differs from natural hormone in benzyl, substituting for an iodine atom in the 3′ position. The benzyl is too large to fit into the enclosed pocket of the receptor. The crystal structure of human thyroid hormone receptor β at 2.8-Å resolution with GC-24 bound explains its agonist activity and unique isoform specificity. The benzyl of GC-24 is accommodated through shifts of 3–4 Å in two helices. These helices are required for binding hormone and positioning the critical helix 12 at the C terminus. Despite these changes, the complex associates with coactivator as tightly as human thyroid hormone receptor bound to thyroid hormone and is fully active. Our data suggest that increased specificity of ligand recognition derives from creating a new hydrophobic cluster with ligand and protein components.

Induction of Larval Tissue Resorption in Xenopus laevis Tadpoles by the Thyroid Hormone Receptor Agonist GC-1

A major challenge in understanding nuclear hormone receptor function is to determine how the same ligand can cause very different tissue-specific responses. Tissue specificity may result from the presence of more than one receptor subtype arising from multiple receptor genes or alternative splicing. Recently, high affinity analogs of nuclear receptor ligands have been synthesized that show subtype selectivity. These analogs can greatly facilitate the study of receptor subtype-specific functions in organisms where mutational analysis is problematic or where it is desirable for receptors to be expressed in their normal physiological contexts. We describe here the effects of the synthetic thyroid hormone analog GC-1 on the metamorphosis of the frog Xenopus laevis. The most potent natural thyroid hormone, 3,5,3′-triidothyronine or T3, shows similar binding affinity and transactivation dose-response curves for both thyroid hormone receptor isotypes, designated TRα and TRβ. GC-1, however, binds to and activates TRβ at least an order of magnitude better than it does TRα. GC-1 efficiently induces death and resorption of premetamorphic tadpole tissues such as the gills and the tail, two tissues that strongly induce thyroid hormone receptor β during metamorphosis. GC-1 has less effect on the growth of adult tissues such as the hindlimbs, which express high TRα levels. The effectiveness of GC-1 in inducing tail resorption and tail gene expression correlates with increasing TRβ levels. These results illustrate the utility of subtype selective ligands as probes of nuclear receptor function in vivo.

Improved synthesis of the iodine-free thyromimetic GC-1.

Synthesis of the TRbeta-selective thyromimetic GC-1 has been improved using methoxymethyl (MOM) and triisopropylsilyl (TiPS) substituents as phenolic protecting groups. The new synthetic route is adaptable to analogue design.

Design, Synthesis, and Evaluation of Thyronamine Analogues as Novel Potent Mouse Trace Amine Associated Receptor 1 (mTAAR1) Agonists.

Trace amine associated receptor 1 (TAAR1) is a G protein coupled receptor (GPCR) expressed in brain and periphery activated by a wide spectrum of agonists that include, but are not limited to, trace amines (TAs), amphetamine-like psychostimulants, and endogenous thyronamines such as thyronamine (T0AM) and 3-iodothyronamine (T1AM). Such polypharmacology has made it challenging to understand the role and the biology of TAAR1. In an effort to understand the molecular basis of TAAR1 activation, we rationally designed and synthesized a small family of thyronamine derivatives. Among them, compounds 2 and 3 appeared to be a good mimic of the parent endogenous thyronamine, T0AM and T1AM, respectively, both in vitro and in vivo. Thus, these compounds offer suitable tools for studying the physiological roles of mouse TAAR1 and could represent the starting point for the development of more potent and selective TAAR1 ligands.

An efficient substitution reaction for the preparation of thyroid hormone analogues

The substitution of the sterically hindered carbon of the potent thyroid hormone agonist, GC-1, was effected by a reaction based on the solvolysis of the benzylic hydroxyl group. The reaction was found to proceed in high yield with a variety of nucleophiles including alcohols, thiols, allyl silanes and electron-rich aromatic compounds, providing a convenient route to the synthesis of new thyroid hormone analogues.

Dual Inhibition of PDK1 and Aurora Kinase A: An Effective Strategy to Induce Differentiation and Apoptosis of Human Glioblastoma Multiforme Stem Cells

The poor prognosis of glioblastoma multiforme (GBM) is mainly attributed to drug resistance mechanisms and to the existence of a subpopulation of glioma stem cells (GSCs). Multitarget compounds able to both affect different deregulated pathways and the GSC subpopulation could escape tumor resistance and, most importantly, eradicate the stem cell reservoir. In this respect, the simultaneous inhibition of phosphoinositide-dependent kinase-1 (PDK1) and aurora kinase A (AurA), each one playing a pivotal role in cellular survival/migration/differentiation, could represent an innovative strategy to overcome GBM resistance and recurrence. Herein, the cross-talk between these pathways was investigated, using the single-target reference compounds MP7 (PDK1 inhibitor) and Alisertib (AurA inhibitor). Furthermore, a new ligand, SA16, was identified for its ability to inhibit the PDK1 and the AurA pathways at once, thus proving to be a useful tool for the simultaneous inhibition of the two kinases. SA16 blocked GBM cell proliferation, reduced tumor invasiveness, and triggered cellular apoptosis. Most importantly, the AurA/PDK1 blocker showed an increased efficacy against GSCs, inducing their differentiation and apoptosis. To the best of our knowledge, this is the first report on combined targeting of PDK1 and AurA. This drug represents an attractive multitarget lead scaffold for the development of new potential treatments for GBM and GSCs.

The TRβ-selective agonist, GC-1, stimulates mitochondrial oxidative processes to a lesser extent than T3

Specific tissue responses to thyroid hormone are mediated by the hormone binding to two subtypes of nuclear receptors, TRalpha and TRbeta. We investigated the relationship between TRbeta activation and liver oxidative metabolism in hypothyroid rats treated with equimolar doses of triiodothyronine (T(3)) and GC-1, a TRbeta agonist. T(3) treatment produces increases in O(2) consumption and H(2)O(2) production higher than those elicited by GC-1. The greater effects of T(3) on oxidative processes are linked to the higher hormonal stimulation of the content of respiratory chain components including autoxidizable electron carriers as demonstrated by the measurement of activities of respiratory complexes and H(2)O(2) generation in the presence of respiratory inhibitors. It is conceivable that these differential effects are dependent on the inability of GC-1 to stimulate TRalpha receptors that are likely involved in the expression of some components of the respiratory chain. The greater increases in reactive oxygen species production and susceptibility to oxidants exhibited by mitochondria from T(3)-treated rats are consistent with their higher lipid and protein oxidative damage and lower resistance to Ca(2)(+) load. The T(3) and GC-1 effects on the expression levels of nuclear respiratory factor-1 and -2 and peroxisome proliferator-activated receptor-gamma coactivator-1alpha suggest the involvement of respiratory factors in the agonist-linked changes in mitochondrial respiratory capacities and H(2)O(2) production.

Cardioprotection by ranolazine in perfused rat heart

Abstract: We used the isolated working rat model to evaluate the effect of therapeutic concentrations (5–10 &mgr;M) of ranolazine on contractile performance, oxygen consumption, irreversible ischemic injury, and sarcoplasmic reticulum (SR) function. Ischemic injury was induced by 30 minutes of global ischemia followed by 120 minutes of Langendorff reperfusion and evaluated on the basis of triphenyltetrazolium chloride staining. SR function was determined on the basis of [3H]-ryanodine binding, the kinetics of calcium-induced calcium release, measured by quick filtration technique, and oxalate-supported calcium uptake. In working hearts, ranolazine significantly reduced oxygen consumption (P = 0.031), in the absence of significant changes in contractile performance, and decreased irreversible ischemic injury (P = 0.011), if administered either before ischemia–reperfusion (25.4% ± 4.7% vs. 42.7% ± 6.0%) or only at the time of reperfusion (20.2% ± 5.2% vs. 43.7% ± 9.9%). In SR experiments, treatment with ranolazine determined a significant reduction in [3H]-ryanodine binding (P = 0.029), because of decreased binding site density (369 ± 9 vs. 405 ± 12 fmol/mg), and in the kinetics of SR calcium release (P = 0.011), whose rate constant was decreased, whereas active calcium uptake was not affected. Ranolazine effectiveness at reperfusion and its ability to module SR calcium release suggest that this drug might be particularly useful to induce cardioprotection during coronary revascularization interventions, although the relevance of the effects on calcium homeostasis remains to be determined.

Effects of Zofenopril on Cardiac Sarcoplasmic Reticulum Calcium Handling

Isolated rat hearts were perfused for 120 minutes in the presence or in the absence of 10 μM zofenoprilat, the active metabolite of zofenopril. At the end of perfusion, cardiac tissue was used to assay sarcoplasmic reticulum (SR) 45Ca uptake and SR calcium release, which was determined by automatized quick filtration technique after SR vesicle loading with 45Ca. The expression of genes involved in the control of calcium homeostasis was evaluated by polymerase chain reaction after reverse transcription. In chronic experiments, SR 45Ca uptake and gene expression were measured in hearts derived from rats treated with 15 mg·kg−1·day−1 zofenopril for 15 days. Acute or chronic zofenopril administration did not produce any change in contractile performance. In acute experiments, SR 45Ca uptake was significantly increased after exposure to zofenoprilat. The rate constant of calcium-induced calcium release was slightly although not significantly higher, and the calcium leak measured under conditions promoting SR channel closure was significantly increased. In the chronic model, significant increase in the rate of SR 45Ca uptake was confirmed. Gene expression was not modified, except for decreased phospholamban expression, which is observed in the acute but not in the chronic model. In conclusion, zofenopril increases SR calcium cycling and stimulates active calcium uptake into the SR.